Neutravidin protein

Biotinylation assays were performed as previously described, with modifications by Acuña et al., 2013 (link). Briefly, cells (1 × 10⁸) were labelled with Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, Rockford, IL, USA). Cells were washed three times in ice-cold PBS 1X Ca⁺⁺/Mg⁺⁺ (pH 7.4) to remove any contaminating proteins and 1mM of Sulfo-NHS-LC-Biotin diluted in ice-cold PBS 1X Ca⁺⁺/Mg⁺⁺ (pH 7.4) was added per total reaction volume. Cells labelled with biotin were incubated at 4 °C for 30 min, washed three times with ice-cold PBS 1X Ca⁺⁺/Mg ⁺⁺ (pH 7.4) and the free biotin was blocked with a solution of 50 mM Tris-HCl and again washed three times with ice-cold PBS 1X Ca⁺⁺/Mg⁺⁺ (pH 7.4). The labelled cells were homogenised in 200 µl of buffer RIPA with Triton X-100 and protease inhibitors and then precipitated by continuous mixing with 40 µl of immobilised NeutrAvidin Protein (Thermo Fisher Scientific, Rockford, IL, USA) for 4 hours at 4 °C. The precipitates were washed four times in ice-cold PBS 1X Ca⁺⁺/Mg⁺⁺ (pH 7.4). The resin-bound complex was boiled in SDS-PAGE sample buffer. Samples were resolved by 10% (w/v) SDS– polyacrylamide gels and Western blot analysis was performed.

Poly lactic-co-glycolic acid (50:50 and 75:25 PLGA) was acquired from Evonik Industries. Poly ethylene-maleic acid (PEMA), poly vinyl alcohol (PVA), chitosan, Dulbecco’s phosphate buffered saline (DPBS), and N-(3-Dimethylaminopropyl)-N-ethyl carbodiimide (EDAC) were purchased from Sigma–Aldrich. DiO and NeutrAvidin protein were acquired from Thermo Fisher. Sialyl Le^a-PAA-biotin (SLe^a) was acquired from GlycoTech. Cutaneous lymphocyte-associated antigen (CLA-PE) was obtained from Miltenyi Biotec. Fluorescein rabbit antimouse IgG-1 was purchased from Jackson Immunoresearch. MESF calibration bead was purchased from Bangslab. IL-1β was acquired from Fitzgerald. Human umbilical vein endothelial cells (HUVECs) and endothelial growth medium-2 (EGM-2 medium) were purchased from Lonza. Colorimetric cell counting kit-8 (CCK-8) reagent was obtained from Dojindo Molecular Technologies. All reagent grade organic solvents were purchased from VWR.

Lyophilized lipids were purchased from Avanti lipids; SUVs were made. Cyclic RGD was purchased from Peptide international (No. PCI-3895-PI). NeutrAvidin Protein, Dylight 650 was purchased from Thermofisher Scientific (No. 84607). To assemble RGD-functionalized supported lipid bi-layers, DOPC was doped with 1,2-dipalmitoylsn-glycero-3-phosphoethanolamine-N-cap biotinyl (16/0 Biotinyl Cap PE) on clean coverslips (No.1.5). These were functionalized with biotin RGD using Dylight 650-Neutravidin as a linker between biotin on the lipid and RGD^{53 (link)}.

Iron (III) chloride hexahydrate was purchased from Across (99%) and sodium oleate from TCI America (97%). Neutravidin protein, Alexa Fluor 647-NHS ester and Biotin-NHS ester were purchased from Thermo Fisher Scientific. LOR photoresist and MF-319 developer were obtained from Microchem and S1805 photoresist was obtained from Microposit. Other reagents and chemicals were purchased from Sigma-Aldrich and used as received unless otherwise stated.